The present invention generally relates to therapeutic strategies utilizing proteinacious channels in lipid membranes of mammalian cells. More particularly, this invention relates to the utilization of the electrophysiology of Kv7 potassium channels and/or L-type calcium channels in vascular smooth muscle cells (VSMCs) and airway smooth muscle cells (ASMCs), and to the use thereof to identify new pharmaceuticals that may be used to treat cardiovascular and airway conditions, including hypertension, stroke and asthma, and to perform drug screening to assess potential risk of pharmaceuticals.
Mammalian cells, including the smooth muscle cells (SMCs) within the walls of arteries (vascular smooth muscle cells, or VSMCs) and the bronchioles of the lung (airway smooth muscle cells, or ASMCs), are surrounded by a lipid membrane which functions as a barrier to diffusion of many soluble substances, including ions, into and out of the cytosol of the cells. Proteinacious channels integrated into these lipid membranes allow ions to cross the lipid membrane when the channels are open. A portion of these proteinacious channels is selective for potassium ions (K+), and are referred to as potassium channels or K+ channels. Still other proteinacious channels are selective for calcium ions (Ca2+), and are referred to as calcium channels or Ca2+ channels. Under normal circumstances, potassium ions (K+) are typically present inside the cell at concentrations about twenty-five times higher as compared to their corresponding concentration outside the cell. When these potassium channels open (activate), potassium ions (K+) tend to leak out of the cell through these potassium channels, resulting in a measurable electrical current across the membrane. This electrical current establishes an electrical charge difference across the lipid membrane (membrane voltage, or Vm), resulting in the polarization of the membrane. Polarization of the membranes of smooth muscle cells has a profound effect on the function of voltage-sensitive L-type Ca2+channels in these cells.
VSMCs and ASMCs are able to contract or relax to regulate blood flow (and blood pressure) and airway resistance, respectively. The contractile state of SMCs is determined by the cytosolic Ca2+ concentration ([Ca2+]cyt). Elevation of [Ca2+]cyt triggers SMC contraction to produce vasoconstriction or airway constriction. Influx of Ca2+through L-type Ca2+channels is a major mechanism for elevation of cytosolic [Ca2+] in SMCs and this depends on membrane voltage (Vm). As noted above, K+ channels represent a primary effector for adjusting Vm. K+ channels of the Kv7 family (KCNQ) were recently identified among the cohort of vascular ion channels. These Kv7 channels, which were previously recognized as mediators of acetylcholine-induced neuronal excitation, have distinctive electrophysiological characteristics: activation at voltages negative to −50 mV, outward rectification, and absence of time-dependent inactivation.
Because polarization of the membranes of SMCs has a profound effect on the function of voltage-sensitive L-type Ca2+ channels in these cells, cell membrane polarization is also a primary determinant of the extent to which arteries and bronchioles constrict or dilate. KCNQ voltage-activated K+ channels (also known as the Kv7 family) play an important role in regulating the membrane voltage of many excitable tissues. See, for example, Delmas et al., “Pathways modulating neural KCNQ/M (Kv7) potassium channels,” Nat Rev Neurosci 6(11):850-862 (2005); and Robbins et al., KCNQ potassium channels: physiology, pathophysiology, and pharmacology,” Pharmacol Ther 90(1):1-19 (2001). Recently, KCNQ5 (Kv7.5) channels were determined to be expressed and functional in vascular smooth muscle cells.
Cyclooxygenase-2 (COX-2) inhibitors are important members of the family of non-steroidal anti-inflammatory drugs (NSAIDs). Celebrex® (celecoxib) and Vioxx® (rofecoxib) were introduced in 1999 and rapidly became frequently prescribed for clinical use as analgesic/anti-inflammatory agents because they prevent the generation of prostaglandins involved in inflammation and pain, while sparing the beneficial effects of cyclooxygenase-1 (COX-1)-generated prostanoids. However, COX-2 inhibitors have been under intense scrutiny since 2004 when Vioxx® was voluntarily withdrawn from the market because of a reported increased risk of myocardial infarction and stroke in patients taking the drug for prolonged periods of time.
A systematic review of randomized clinical trials of COX inhibitors revealed that rofecoxib, a highly COX-2-selective agent, and diclofenac, an NSAID with COX-2/COX-1 selectivity similar to celecoxib, both significantly increased the risk of cardiovascular (CV) events. In contrast, a number of clinical studies failed to demonstrate an increased CV risk with celecoxib relative to placebo. See, for example, McGettigan et al., “Cardiovascular Risk and Inhibition of Cyclooxygenase: A Systematic Review of the Observational Studies of Selective and Nonselective Inhibitors of Cyclooxygenase 2,” Journal of the American Medical Association 296:1633-1644 (2006), and White et al., “Risk of Cardiovascular Events in Patients Receiving Celecoxib: A Meta-Analysis of Randomized Clinical Trials,” The American Journal of Cardiology 99(1):91-98 (2007). The reasons for the differences between celecoxib and other COX-2 inhibitors have been widely debated.
HERG (human ether-a-go-go related gene) encodes a particular type of potassium channel (Kv11.1) that contributes to the electrical activity of the heart. To avoid unwanted cardiac side effects, new drugs in development are commonly screened for effects on Kv11.1 potassium channel currents using cultured cells engineered to express large numbers of these channels. In contrast, smooth muscle Kv7 channels have not been recognized as a potential site of adverse (or beneficial) drug action and therefore no airway or vascular smooth muscle Kv7 channel screening assays have been developed. Prior to a recent report (Brueggemann et al., “Differential Effects of Selective COX-2 Inhibitors on Vascular Smooth Muscle Ion Channels May Account for Differences in Cardiovascular Risk Profiles,” Molecular Pharmacology 76: 1053-1061 (2009)), COX inhibitors had not been reported to exert any effects on vascular smooth muscle Kv7 channels or vascular smooth muscle L-type Ca2+ channels, and therefore no therapeutic strategies have been proposed to use these drugs to treat vasospasm or bronchospasm that can lead to heart attacks and strokes or airway obstruction, respectively.
HERG channel screening assays do not detect effects of drugs on smooth muscle Kv7 channel activity and therefore are not useful for predicting potential adverse cardiovascular side effects associated with such activity or for predicting potential beneficial therapeutic effects associated with such activity.